Wilsonomyces Carpophilus Research Articles

New secondary metabolites produced by the phytopathogenic fungus Wilsonomyces carpophilus

Two new metabolites possessing the unusual 1-oxa-7-azaspiro[4.4]non-2- ene-4,6-dione core (2, 3) along with the recently described pseurotin A3 (1) were isolated from the pathogenic fungus Wilsonomyces carpophilus(previously named Stigmina carpophila). The producer organism was obtained from Prunus armeniaca collected in Iran and was identified by morphological and molecular phylogenetic methods. The structures of the isolated compounds were elucidated on the basis of extensive NMR spectroscopic analysis, high-resolution mass spectrometry and ECD analysis. The compounds were screened for their antimicrobial, cytotoxic, nematicidal and biofilm inhibition activities but, no significant effect was observed. To the best of our knowledge, this is the first report on the isolation of secondary metabolites produced by W. carpophilus.

Morpho-cultural, pathological and molecular variability in Thyrostroma carpophilum causing shot hole of stone fruits in India

Shot hole disease of stone fruits caused by Thyrostroma carpophilum has become a major threat to stone fruit industry of Jammu and Kashmir, India because of the failure in its management with fungicides. To understand the diversity in shot hole pathogen, a combination of conventional (morphological, cultural and pathological) and molecular (ISSR and ITS markers) approaches were employed to discern variability in 25 isolates of T. carpophilum isolated from peach, plum, apricot, almond and cherry leaves collected from Srinagar, Ganderbal, and Baramulla districts of Jammu and Kashmir, India. The studies revealed a high level of variability among the pathogen. Based on the morpho-cultural and pathological studies, the isolates were grouped into different categories based on colony growth, texture, margin and colour besides change in media colour, incubation period, leaf area infected, etc. Using ISSR markers, a high level of polymorphism in different isolates of T. carpophilum was observed which indicated that these markers are suitable for studying the genetic diversity in this pathogen. Based on dendrogram, the isolates were grouped irrespective of their geographical origin or host species. Phylogenetic analysis of the 25 sequences based on ITS region showed maximum similarity with T. carpophilum (Syn. Wilsonomyces carpophilus) sequences retrieved from NCBI and grouped them in a single clade which proved it as a powerful tool for authentic identification. The pathogen was highly variable based on morpho-cultural, pathological and molecular (ISSR) characterisation.

Comparing disease resistance of local and international plum cultivars (Prunus domestica) from Eastern Transylvania, Romania

Assessments were carried out to evaluate disease susceptibility in two local and three internationally recognised plum cultivars (‘D’Agen’, ‘Stanley’ and ‘Tuleu gras’). The following pathogens were evaluated weekly: plum pox virus (PPV), funguses blackthorn dotty (Polystigma rubrum), red rust (Tranzscheila spp.), shot-hole disease (Stigmina carpophila) and blossom blight (Monilinia laxa). According to the results, one of the local cultivars assessed (Local 2) presented virtually no virus infestation even under high mealy plum aphid (Hyalopterus pruni) density. Therefore, this cultivar can be considered PPV resistant. Local cultivar 1 also presented very low susceptibility to shot hole and blossom blight. Both may be promising genetic sources in breeding programmes against pathogens.

Almond diseases and disorders

Almond production has become a major horticultural industry in Australia over the last ten years. Due to the relatively recent expansion of the industry and limited published information on local diseases and disorders, overseas literature was initially relied on to aid diagnosis of tree health issues. As knowledge of the diseases of almonds in Australia increases, this information will become more relevant as a reference for local growers and researchers. A range of diseases have significant impacts on almond production. Symptoms and key features of major diseases of almonds are described under the categories: foliar or fruit; and trunk, crown and root diseases. The diseases under the respective categories are: hull rot (Rhizopus spp.), bacterial spot (Xanthomonas arboricola pv. pruni), rust (Tranzschelia discolor), shot-hole (Wilsonomyces carpophilus), almond scab (Cladosporium carpophilum); silver leaf (Chondrostereum purpureum), Verticillium wilt (Verticillium dahliae), and trunk and root rot (Phytophthora and Fusarium spp.). Certain foliar disease symptoms are similar to either nutritional or physiological disorders or pesticide toxicity. Correct diagnosis of diseases and disorders is crucial for development and implementation of appropriate disease management strategies.

The Response of Several Plum Cultivars to Natural Infection with <i>Monilinia laxa, Polystigma rubrum </i>and<i> Stigmina carpophila</i>

The response to the attack of Monilinia laxa, Polystigma rubrum and Stigmina carpophila in natural conditions of infection of 13 plum cultivars were evaluated during three years, in a commercial orchard located in North-western of Romania. The studied cultivars were: ‘Topfirst’, ‘Nectarina rosie’, ‘Tuleu timpuriu’, ‘Hangata’, ‘Toptaste’, ‘Tuleu gras’, ‘Vinete de Italia’, ‘Stanley’, ‘Vinete romanesti’, ‘Tophit’, ‘Jojo’,‘Anna Späth’, ‘Topend’. The highest degree of flower attack by Monilinia laxa was recorded for ‘Stanley’, while the lowest attacks were registered on ‘Topend’, ‘Jojo’, ‘Tophit’ and ‘Nectarina rosie’. The best response regarding the attack on fruits proved to be with ‘Topend’, ‘Jojo’, ‘Tophit’ and ‘Topfirst’. These cultivars registered small values of attack degree (0.7-3.3%). The cultivars with the lowest attack degree of red staining caused by Polystigma rubrum were ‘Top End’, ‘Jojo’, ‘Tophit’, ‘Toptaste’, ‘Nectarina rosie’ and ‘Topfirst’. A high degree of attack was calculated on ‘Anna Späth’, ‘Vinete romanesti’, ‘Vinete de Italia’ and ‘Tuleu timpuriu’. These cultivars prove to be very sensitive to the red staining disease in natural conditions of infections. Cultivars ‘Top End’, ‘Jojo’, ‘Tophit’, ‘Toptaste’, ‘Nectarina rosie’ and ‘Topfirst’ were slightly attacked by Stigmina carpophila (5.3-7.3% attack degree). The low infections levels recommend the mentioned cultivars as possible genitors in plum breeding programs, for creating new genotypes with good response to diseases attack.

The Response of Several Plum Cultivars to Natural Infection with <i>Monilinia laxa, Polystigma rubrum </i>and<i> Stigmina carpophila</i>

The response to the attack of Monilinia laxa, Polystigma rubrum and Stigmina carpophila in natural conditions of infection of 13 plum cultivars were evaluated during three years, in a commercial orchard located in North-western of Romania. The studied cultivars were: ‘Topfirst’, ‘Nectarina rosie’, ‘Tuleu timpuriu’, ‘Hangata’, ‘Toptaste’, ‘Tuleu gras’, ‘Vinete de Italia’, ‘Stanley’, ‘Vinete romanesti’, ‘Tophit’, ‘Jojo’,‘Anna Späth’, ‘Topend’. The highest degree of flower attack by Monilinia laxa was recorded for ‘Stanley’, while the lowest attacks were registered on ‘Topend’, ‘Jojo’, ‘Tophit’ and ‘Nectarina rosie’. The best response regarding the attack on fruits proved to be with ‘Topend’, ‘Jojo’, ‘Tophit’ and ‘Topfirst’. These cultivars registered small values of attack degree (0.7-3.3%). The cultivars with the lowest attack degree of red staining caused by Polystigma rubrum were ‘Top End’, ‘Jojo’, ‘Tophit’, ‘Toptaste’, ‘Nectarina rosie’ and ‘Topfirst’. A high degree of attack was calculated on ‘Anna Späth’, ‘Vinete romanesti’, ‘Vinete de Italia’ and ‘Tuleu timpuriu’. These cultivars prove to be very sensitive to the red staining disease in natural conditions of infections. Cultivars ‘Top End’, ‘Jojo’, ‘Tophit’, ‘Toptaste’, ‘Nectarina rosie’ and ‘Topfirst’ were slightly attacked by Stigmina carpophila (5.3-7.3% attack degree). The low infections levels recommend the mentioned cultivars as possible genitors in plum breeding programs, for creating new genotypes with good response to diseases attack.

Perspectives of breeding fungal diseases resistant cultivars of nectarine (Prunus persica (L.) Batsch. subsp. nectarina (Ait.) Shof.)

There are allocated some forms of distant hybrids with weak degree of fungal diseases lesion (Taphrina deformans (Berk.) Tul., Sphaerotheca pannosa (Lev.) var. persicae Woron., Clasterosporium carpophilum Aderh., Monilia cinerea Bonord. и Monilia fructigena Fr.) that are of practical interest for breeding such cultivars of peach and nectarine that will resist these pathogens.

Effect of a plastic rain shield on fruit cracking and cherry diseases in Greek orchards

The use of plastic rain shields is a very effective method to control fruit cracking in cherry trees. In this study, the effect of plastic rain shields on fruit cracking and fruit decay was investigated in different sweet cherry cultivars. The results showed that the percentage of fruit cracking and fruit decay was significant higher in uncovered than covered cherry trees. Fungi of the genus Monilinia were the main pathogens causing fruit decay in cherry fruits. In fruit with symptoms of cracking, fungi of the genus Alternaria predominated. Finally, the effect of plastic rain shield on the development of the disease Shot Hole (Wilsonomyces carpophilus) was examined. In most of cases, the percentage of the Shot Hole disease was higher in covered than uncovered cherry trees. Differences were found among cultivars.Generally, the use of plastic rain shields is a very effective method to control fruit cracking and fruit decay on cherry trees, but they should be removed during dry periods to reduce the risk of developing of Shot Hole.

Study on morphology, pathogenicity and genetic diversity of Wilsonomyces carpophilus isolates, the causal agent of shot hole of stone fruit trees based on RAPD-PCR in Iran

Shot hole disease is one of the most important diseases of stone fruit trees in Iran. The disease is wide spread among orchards of Prunus spp. During spring and summer of 2007, 80 monoconidial isolates of the pathogen were recovered from infected leaves, fruits and twigs of different Prunus spp. in West Azerbaijan, Tehran, Ghazvin and Razavi Khorasan provinces of Iran and were studied taxonomically. Based on morphological and physiological characteristics and growth optimal temperature, all isolates were identified as Wilsonomyces carpophilus. Seedlings of stone fruits (apricot, almond, peach, nectarine, plum, sweet cherry and sour cherry) were used for pathogenicity tests. All seedlings were susceptible to the fungal isolates and showed disease symptoms on twigs, leaves, buds and petioles. Genetic diversity of 28 selected fungal isolates was investigated based on DNA fingerprinting by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), using four random primers. Based on cluster analysis of the PCR results from the four primers, 10 fingerprinting groups (clonal lineages) and 27 haplotypes were identified. Clonal lineages “C”, “D” and “E”, each with six haplotypes formed the biggest clonal lineages, but other clonal lineages (“B”, “F”, “G”, “H”, “I” and “J”) included only one isolate. No correlation was detected among clonal lineages with the location of selected isolates and their host species. A correlation was found between the substrate (fruit, twig or leaf) and clonal lineages, particularly in “C” clonal lineage. The results showed that the fungus population had high genetic diversity which is distributed among the different areas of Iran.

Field assessment of vulnerability to fungi diseases of varieties and forms of nectarine (Prunus persica (L.) Batsch. subsp. nectarina (Ait.) Shof.) displaying male sterility characteristic from Niktskyi Botanic Garden collection

The varieties and forms of nectarine resistant to fungi diseases (Clasterosporium carpophilum (Lev.) Aderh., Taphrina deformans Tul., Sphaerotheca pannosa (Wallr.) Fr. Lev. var. persicae Woronich) having the practical interest for modern technology growing and for breeding on these characteristics have been selected.

Selection of autochthonous sour cherry (Prunus cerasus L.) genotypes in Feketic region

Autochthonous genotypes of fruit species are very important source of genetic variability and valuable material for breeding work. Fruit Research Institute-Cacak has a long tradition of studying autochthonous genotypes of temperate fruits sporadically spread and preserved in some localities in Serbia. Over 2005-2006, the following properties of nine autochthonous sour cherry genotypes grown in Feketic region were investigated: flowering and ripening time, pomological properties, biochemical composition of fruits and field resistance to causal agents of cherry diseases - cherry leaf spot (Blumeriella jaapii (Rehm.) v. Arx.), shot-hole (Clasterosporium carpophilum (L?v.) Aderh.) and brown rot (Monilinia laxa /Ader et Ruhl./ Honey ex Whetz.). The genotypes were tested for the presence of Prune dwarf virus and Prunus necrotic ring spot virus. In majority of genotypes fruits were large, with exceptional organoleptical properties, whereas ripening time was in the first ten or twenty days of June. The highest fruit weight was observed in F-1 genotype (8.1 g). The highest soluble solids and total sugars content were found in F- 4 genotype (17.60% and 14.25%, respectively). As for field resistance to causal agents of diseases and good pomo-technological properties, F-1, F-2, F-3, F-7 and F-8 genotypes were singled out.

The perspectives of using hybrids between nectarine (Prunus persica (L.) Batch subsp. nectarina (Ait. Shof.) and almond (Prunus amygdalus Batsch) in selection of varietes and rootstocks

The perspective hybrid forms between genus Prunus persica (L.) Batch and Prunus amygdalus Batsch, having high ornamental qualities and high resistance to Sphaerotheca pannosa (L v.) var. persicae Woronich., Taphrina deformans (Berk.) Tul., Clasterosporium carpophilum (L. v) Aderh., Monilia cinerea Bonord. have been obtained for using in breeding of peach and nectarine varieties and their rootstocks.

A new fungicide based on copper with low concentration and high dispersion for plum, sweet cherry and sour cherry.

Copper sulphate remains an important fungicide for combating major diseases in stone fruits species. Using of this fungicide repeatedly creates some problems such as accumulation of copper ions in the soil until it becomes toxic concentrations for plant and micro flora and micro fauna of the soil. This paper presents results of a study verifying the effectiveness of copper sulphate as fungal substance applied in doses of 18 to 25 times smaller than conventional commonly used substances such as Bordelaise mixture or Kocide 101WP. In this formulation, the cooper has high dispersion made by depositing it on a bioactive support. The bioactive support contains the following ions: Mg2+, PO43-, Ca2+, NH4+ which represent food source for the trees. In the same time, this formulation are both part of fungicide and foliar fertilizer. This Cooper formula proved to be very effective against the most important diseases of plum (Monilinia laxa, Xanthomonas pruni, Polistigma rubrum, Taphrina pruni) sweet cherry and sour cherry (Pseudomonas syringae van Hall, Coccomyces hiemalis Higg, Stigmina carpophila (Lev.) M.B. and Monilinia laxa (Aderh et Ruhl) Hon Ellis), the attack degree two or three time have been reduced. Cooper HD (high dispersion) can be administered together with a series of leaf fertilizers situation where it is absorbed by the surface of leaves it possesses an effect of longer duration. Lack of alkaline reaction makes it compatible with the majority of pesticides. The advantages it displays recommends this copper formula for the technologies of phyto-sanitary protection of the stone-fruits.

Field reactions of plum cultivars to the shot-hole disease in southern Italy

The reactions to shot-hole disease ( Stigmina carpophila) of nine plum cultivars and seed-propagated myrobalan trees were evaluated during three years in three commercial orchards located at Nova Siri, Matera, Italy. In July 2006, May 2007, June 2007 and June 2008, shot-hole severity and twig defoliation were assessed. Pathogen pressure was higher in 2007 and 2008 than in 2006. In the three years of study, cultivar Golden Plumza was the most susceptible to the shot-hole measured by leaf severity, however it showed variable defoliation percentages in different years. The seed-propagated myrobalan trees were the most resistant, although they showed a slight degree of defoliation in 2007 and 2008. Intermediate reactions to the disease occurred in cultivars Angeleno, Autumn Giant, Fortune, Green Sun, October Sun, Santa Rosa, Sorriso di Primavera and T.C. Sun. A significant correlation between shot-hole leaf severity and twig defoliation was detected. To our knowledge, this is the first report on screening for resistance of plum cultivars to shot-hole disease.

Promising sour cherry hybrids (Prunus cerasus L.) developed at Fruit Research Institute Cacak

At Fruit Research Institute in Cacak, major objectives of the work on breeding new sour cherry (Prunus cerasus L.) cultivars are high cropping, large, high-quality fruits and resistance to causal agents of diseases and pests. As a result of the planned hybridization, more than 10,000 hybrid seedlings have been developed from about 40 cultivars within more than 110 parental combinations, among which are 'Cacanski rubin' ('Shasse Morello' x 'K?r?ser Weichsel') and 'Sumadinka' ('K?r?ser Weichsel' x 'Heimanns Konserven Weichsel') which have been named and released. Ten-year study of 11 hybrids, selected from the population of about 3,000 hybrid seedlings, gave four hybrids which have been singled out as elite (III/23, III/31, II/40 i XII/57). These hybrids are currently under procedure of being released as new cultivars. The paper presents two-year results of the study of ripening time, pomological properties, biochemical composition of fruits, and field resistance to causal agents of diseases and pests attacking the above named genotypes which were compared to standard cultivar 'Heimanns Konserven Weichsel'. In the studied hybrids, fruit weight, soluble solids content and sugars content were higher than in standard cultivar. In addition, they exhibit substantial field resistance to causal agents of brown rot (Monilinia laxa /Ader et Ruhl./ Honey ex Whetz.), cherry leaf spot (Blumeriella jaapii (Rehm.) v. Arx.), shot-hole (Clasterosporium carpophilum (L?v.) Aderh.) and cherry fruit fly (Rhagoletis cerasi L.) attack.

Isolation and pathogenicity tests of Iranian cultures of the shot hole pathogen of Prunus species, Wilsonomyces carpophilus

During spring and summer of 2007, 76 fungal isolates were recovered from different Prunus species in West Azerbaijan and Tehran provinces of Iran. Based on morphological characteristics, all isolates were identified as Wilsonomyces carpophilus. The occurrence of shot hole disease symptoms on almond, plum, peach and nectarine fruits, apricot twigs in orchards was observed. The first pathogenicity tests with W. carpophilus in Iran, using seven Prunus species, are reported.

Susceptibility of several Japanese plum cultivars to pests and diseases in a newly established organic orchard

The susceptibility of 14 Japanese plum cultivars to an aphid pest and three fungal diseases was analysed during 2007 and 2008 in two experimental orchards, one in organic and the other in conventional management. There was a significantly higher incidence of green peach aphids [ Myzus persicae (Sulzer)] for most of the cultivars in the organically managed treatment. ‘Fortune’, ‘Primetime’, ‘Larry Ann’ and ‘Sapphire’ were the cultivars most affected, and ‘Souvenir’ suffered the lowest attack in both treatments. Shot hole disease ( Wilsonomyces carpophilus) affected both orchards at low levels, and only two cultivars, ‘Santa Rosa’ and ‘Larry Ann’, suffered a moderate attack. Powdery mildew ( Sphaerotheca pannosa) incidence was significantly higher in the conventionally managed orchard and only the ‘Red Beaut’ cultivar was affected. Rust disease ( Tranzschelia spp.) incidence was significantly higher in the organic treatment. The cultivars in which the rust disease appeared earliest and reached the highest levels were ‘Showtime’, ‘Santa Rosa’, ‘Friar’, and ‘Larry Ann’. An intense rust infestation in leaves provoked a premature defoliation in the organic plot, indicating that proper control of this disease is important to maintain tree vigour and yield.

Copper-ammoniac phosphate in the treatment of certain stone fruits.

A new chemical formula of fungicide based on copper (copper-ammoniac phosphate) was tested between 2007 and 2008 at SC Agroindustriala SA Cluj-Napoca, along with copper based fungicides widely spread and used in the protection of stone fruit species. The fungicides spoken of were: Funguran, Kocide 101WP, Champion 50 WP, CBF (copper based fungicides), which display a similar behaviour with statistically ensured differences in the control of Monilinia laxa, Xanthomonas pruni, Polistigma rubrum, Taphrina pruni in the plum, Monilinia laxa, Coccomyces hiemalis, Stigmina carpophila, Xanthomonas campestris in the cherry and Stigmina carpophila, Monilinia laxa and Coccomyces hiemalis in the sour cherry. The advantages displayed by CBF are: efficiency similar to the acknowledged copper based fungicides with a cooper doses ten times lower per hectare, meaning ten times lower price, long lasting effect, as through the NH4+ grouping they connect the foliar surface, non-polluting clean product, formula compatible with diverse foliar fertilizers. Thus, this product lowers the degree of attack to 1/3 and 2/3 as to the untreated control, similarly to the acknowledged fungicides.

Doenças foliares, cancro e número de frutos relacionados com a adubação nitrogenada em pessegueiro

Para a implementação da Produção Integrada (PI) de Pêssegos no Paraná foram necessárias algumas adaptações regionais no manejo da cultura. A recomendação de adubação nitrogenada para o Rio Grande do Sul determinava, em 2003, um máximo anual de 80 kg/ha de nitrogênio (N), o que foi considerado um fator limitante para as altas produtividades obtidas por alguns produtores no Estado do Paraná. Por outro lado, o excesso de nitrogênio pode elevar a incidência de doenças de folhas e de ramos, prejudicando o desenvolvimento das plantas. O presente trabalho objetivou avaliar o efeito da adubação nitrogenada na produção e em três doenças de pessegueiro cultivado em sistema de PI. O experimento foi realizado no município da Lapa, com a cultivar Chimarrita, com três tratamentos (40, 80 e 160 kg/ha de N) e seis repetições. A produção foi avaliada contando-se o número de frutos totais por planta útil na colheita. Para avaliação das doenças, foi determinado o número de lesões (cancros) de Botryospheria dothidea em uma planta marcada por parcela em dois anos consecutivos (2003 e 2004), no período pós-floração. Para furo de bala (Wilsonomyces carpophilus) determinou-se a incidência da doença no ano de 2002 e para ferrugem (Tranzschelia discolor) avaliou-se incidência e severidade em 2003 e 2004. Com os dados de severidade da ferrugem no tempo foi obtida a curva de progresso da doença e o índice da desfolha no período. No terceiro ano observou-se que na dose de 160 kg/ha de N o número de frutos foi estatisticamente significativo e maior que nos demais tratamentos. A incidência de cancros aumentou entre os anos, porém não houve diferenças significativas entre as dosagens de nitrogênio, o furo de bala também não foi influenciado pelos tratamentos. A área abaixo da curva de progresso da doença (AACPD) para severidade de ferrugem foi 20,7 % superior na menor dose em relação a maior dose de nitrogênio, entretanto, sem reflexos sobre a desfolha.

Elaboração de escala diagramática para furo-de-bala e avaliação de doenças foliares em dois sistemas de produção de pessegueiro

As principais doenças foliares que ocorrem em pessegueiro, em Lapa e Araucária, municípios do Estado do Paraná (Brasil), são a ferrugem (Tranzschelia discolor) e o furo-de-bala (Wilsonomyces carpophilus). A primeira causa desfolha logo após a colheita, e a segunda está sempre presente nos pomares, entretanto seus efeitos não são claramente conhecidos e também faltam metodologias para avaliação dessa doença. O presente trabalho objetivou elaborar escala diagramática para furo-de-bala e comparar incidência e severidade de furo-de-bala e da ferrugem em dois sistemas de manejo: produção integrada e convencional. A escala diagramática para furo de bala foi elaborada no primeiro ano de avaliação, selecionando-se folhas com diferentes níveis de severidade, sendo estabelecidos os níveis mínimo e máximo da doença no campo, e os demais níveis foram interpolados de acordo com a acuidade visual. Para comparar os dois sistemas de produção, foram instaladas duas áreas experimentais em duas regiões produtoras (Lapa e Araucária), comparando-se produção integrada (PI) e produção convencional (PC) em duas safras (2002-2003 e 2003-2004), em pomares comerciais de pessegueiro, cultivar 'Chimarrita'. O tratamento PI foi conduzido seguindo as normas técnicas do sistema de PI para pêssego, e o tratamento PC, seguindo o manejo utilizado pelo produtor em cada área. Para avaliar as doenças, foram quantificadas se a incidência e a severidade do furo-de-bala e da ferrugem em ramos marcados, de outubro a abril, nas duas safras. Além disso, foi determinada a desfolha no período de avaliação. A escala diagramática desenvolvida estabeleceu seis níveis de severidade para furo-de-bala, podendo ser indicada para avaliações de comparação entre sistemas e monitoramento da doença no campo. Em Araucária, a incidência e a severidade de furo-de-bala tiveram comportamento inverso entre os anos, sendo menor na PI em primeira safra e maior na segunda, a ferrugem foi menos severa e provocou menor desfolha na PI, no primeiro ano. Em Lapa, não foram observadas diferenças entre os sistemas para furo-de-bala; e para ferrugem, a produção convencional foi melhor, não sendo indicado reduzir pulverizações nesta fase em áreas com alto inóculo do patógeno.